Compositions for regenerating tissue that has deteriorated, and methods for using such compositions

ABSTRACT

The invention provides a composition for promoting regeneration of tissue which has degenerated in a subject as a result of a disease or disorder and a method of using the composition is provided. The composition comprises a biodegradable acellular matrix, and passaged autologous fibroblasts substantially free of immunogenic proteins, e.g., culture medium serum-derived proteins, integrated within the matrix. Also provided is an injectable composition comprising an acellular filler material (e.g., any type of collagen) and passaged autologous fibroblasts substantially free of immunogenic proteins, e.g., culture medium serum-derived proteins, for correcting defects in skin, such as wrinkles or scars, and for augmenting tissue in the subject, particularly facial tissue.

This application is a continuation and claims priority of U.S.application Ser. No. 09/678,047, filed Oct. 3, 2000 now U.S. Pat. No.6,432,710, which is a continuation-in-part and claims priority of U.S.application Ser. No. 09/316,245, filed May 21, 1999, now abandoned,which is a continuation-in-part and claims priority of U.S. applicationSer. No. 09/083,618, filed May 22, 1998, now abandoned. U.S. applicationSer. Nos. 09/678,047, 09/316,245, and 09/083,613 are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

The present invention concerns the regeneration of tissues in a subjectthat have degenerated as a result of a disease or disorder in thesubject. More particularly, the present invention concerns novelcompositions for use in surgical and nonsurgical techniques that promoteregeneration of tissue whose mass has been diminished due to a diseaseor disorder in a subject, correct defects in the skin of subjects, oraugment tissue in subjects. Also disclosed is the use of a novelcomposition in conjunction with a biodegradable acellular matrix forameliorating defects in the tissues, and methods for using the novelcomposition.

BACKGROUND OF THE INVENTION

“Periodontal disease” is the term commonly used to describe inflammatorydisease of the periodontium, i.e., the tissue surrounding and securingteeth to the jawbone. The condition is characterized by inflammatory anddegenerative processes that develop at the gingival margin (gingivitis)and lead to a progressive breakdown and resorption of the periodontalligament and bone (periodontitis), oftentimes resulting in severediminution of the periodontium. Periodontal disease is the leading causeof tooth loss in adults after middle age. [Anderson's Pathology, p.2000, John M. Kissane ed., 9th ed. (1992)].

Periodontal disease results from the accumulation of bacterial plaque inthe gap between the gingiva and the tooth. While anaerobic bacteria arethe primary etiologic agents, the destructive process is believed to bemediated in large part by immunologic reactions of the host. As thedisease progresses, a periodontal pocket is established below thegingival margin, thus prolonging and promoting the inflammatory process.Successive inflammatory reactions result in the progressive erosion ofthe tooth-supporting tissues, i.e., the collagenous fibers making up theperiodontal ligament and the bone pocket in which the tooth sits.[Reviewed in Anderson's Pathology, pp. 1999-2000, John M. Kissane ed.,9th ed. (1992); Shafer et al., A Textbook of Oral Pathology, 4th ed.(1983)].

Periodontal disease can be diagnosed by checking the gingiva forinflammation, probing the depths of periodontal pockets, checkingclinical attachment level, and assessing bone loss by means ofautoradiography. [Jeffcoat, M. K., et al., J. Am. Dent. Assoc.,128:713-724 (1997)].

A number of techniques, both surgical and nonsurgical, have beendeveloped to treat periodontal disease. Particularly with respect tosevere periodontitis, none of the currently available treatments arewholly satisfactory.

For relatively mild cases of periodontitis, practitioners havetraditionally employed nonsurgical mechanical debridement (i.e. scalingand root planing) to remove the bacterial plaque whose accumulationperpetuates the disease, thereby reducing inflammation. Mechanicaldebridement can be accomplished using manual, sonic or ultrasonicinstruments. Scaling and root planing have been shown to decreasegingival inflammation, decrease probe depth, and promote maintenance ofclinical attachment level. However, without resorting to surgicalprocedures, access to root surfaces and bony defects is restricted, andonly limited debridement is possible. [Jeffcoat, M. K., et al., J. Am.Dent. Assoc. 128:713-724 (1997)].

As a result, nonsurgical scaling and root planing is insufficient totreat more severe cases of periodontitis, and it is necessary to resortto more aggressive surgical techniques. Surgical techniques comprisereflecting the gingival tissues to provide access to root surfaces andbone defects, in order that mechanical debridement may be accomplisheddirectly. Following debridement, the gingival tissue is sutured back inposition. Currently available surgical approaches entail substantialpatient discomfort and fail to consistently provide satisfactoryoutcome.

There are a number of non-surgical, non-mechanical approaches totreating periodontal disease, including supragingival and subgingivalirrigation and the application of chemical and antimicrobial agents. Yetnone of these approaches have achieved more than marginal success[Jeffcoat, M. K., et al., J. Am. Dent. Assoc., 128:713-724 (1997)]. Inparticular, there are a number of deleterious side effects associatedwith the use of antibiotics, along with risks such as drug sensitivityand the emergence of antibiotic-resistant pathogens.

Another approach to combating destruction of the periodontal tissue hasfocused on inhibiting the matrix metalloproteases responsible for thisdestruction. Tetracyclines in particular have shown promise asinhibitors of extracellular collagenases, but cause the same sideeffects associated with antibiotics in general. Modified forms oftetracycline have been developed which are non-antimicrobial and retaintheir ability to inhibit collagenases, but these chemically modifiedtetracyclines are not commercially available. [Ciancio, G. C. et al., J.Am. Dent. Assoc. 123:34-43 (1992)].

Because cell proliferation, cell migration and matrix synthesis areprerequisites for periodontal regeneration, some researchers haveattempted to use tissue growth factors, for example insulin-like growthfactor, platelet-derived growth factor, and transforming growth factorto promote periodontal regeneration.

In summary, none of the nonmechanical approaches to treatingperiodontitis have been able to offer more than modest, short termenhancement of traditional mechanical debridement. As noted in a 1997review of techniques used in treating periodontal disease, “scaling androot planning accompanied by oral hygiene procedures remains the firstmode of treatment for adult periodontitis.” [Jeffcoat, M. K. et al., J.Am. Dent. Assoc., 128:713-724 (1997)].

A great deal of research has been directed to methods of regeneratingperiodontal tissue lost as a consequence of periodontal disease, but asyet no wholly satisfactory method is available. For the most part,efforts have focused on surgical approaches that fill the defects with avariety of materials (bone grafting) or use guided tissue regeneration.

Bone grafting techniques involve the use of natural bone or syntheticbone materials. Natural bone grafts are typically either autografts(grafts transferred from one position in the body of a patient toanother position in the body of the same patient) or allografts (graftstransferred from one person to another). Clinicians using natural bonegrafts have had limited success in inducing new bone growth. Problemsassociated with the use of autografts include the need for a secondsurgical site and, in some cases, fresh grafts may be associated withroot resorption. [Jeffcoat, M. K. et al., J. Am. Dent. Assoc.128:713-724 (1997)].

Freeze-dried, demineralized bone has been used as an allograft and shownto promote bone formation. However, the predictability and the amount ofbone fill achieved varies. [Jeffcoat, M. K. et al., J. Am. Dent. Assoc.,128:713-724 (1997)]. Since allografts are transferred from one person toanother, the potential exists that viruses or other pathogens might betransferred to the patient.

Synthetic bone materials which have been investigated include plaster,calcium carbonates, and ceramics such as hydroxyapatite. Clinical trialshave demonstrated that the use of synthetic grafts has resulted inimprovements in probing depth and attachment level. Histologic findings,however, indicate that, in general, synthetic grafts act primarily asspace fillers, with little if any regeneration. [Jeffcoat, M. K. et al.,J. Am. Dent. Assoc., 128:713-724 (1997)].

Guided tissue regeneration is a surgical approach based on placing amembrane barrier under a soft tissue flap above the area of bone loss toenhance wound healing potential. [Ciancio, G. C. et al., J. Am. Dent.Assoc., 123:34-43 (1992]). Investigators have studied both resorbableand nonresorbable membranes. A significant disadvantage of using anonresorbable membrane is the requirement of a second surgical procedureafter approximately six weeks to remove the membrane. Furthermore, inabout 40%-50% of the cases, such membranes become infected in thepatient. While less evidence is available for resorbable membranes thanfor nonresorbable membranes, improvements in clinical attachment levelshave been shown for both types of membranes compared with debridementalone. Most favorable results are reported for Class II furcations inthe mandible and for intrabony defects. Less favorable results have beenreported in maxillary molar and Class III (through and through)furcation defects. [Jeffcoat, M. K. et al., J. Am. Dent. Assoc.,128:731-724 (1997)].

In summary, none of the currently available treatments for periodontaldisease is wholly satisfactory, particularly with regards toregenerating periodontal tissue lost as a result of periodontitis.

The oral mucosa is the tissue lining the oral cavity. There are a numberof conditions that can result in defects in the oral mucosa, for exampletrauma, dermatoses, recurrent aphthous stomatitis, and infections.[Flint, S., The Practitioner 235:56-63 (1991)]. There is currently nowholly satisfactory means of correcting these defects.

Furthermore, there have been efforts to develop and use compositions andmethods to correct defects in skin, such as scars and wrinkles, or toaugment the tissue of a subject in order to improve the appearance ofthe skin, particularly facial skin. The principal method employed tocorrect such defects involves injecting a filler composition into thedermal layer of the skin proximate to the defect or desired tissueaugmentation. Examples of non-biological filler compositions used inthese roles include mineral oil, paraffin, silicone fluid, autologousfat, gelatin powder mixes, polymethylmethacrylate microspheres,cross-linked polydimethylsiloxane, ““TEFLON”” paste, reconstitutedbovine collage, and autologous human collagen.

However, the use of these compositions comprises inherent limitations.For example, the use of mineral oil, paraffin and similar oils and waxeshas resulted in complications such as local chronic edema,lymphadenopathy, scarring and ulcerations (Devore et al., Effectivenessof injectable filler materials for smoothing wrinkle lines and depressedscars. Medical Progress Through Technology 20:243-250 (1994 which ishereby incorporated by reference in its entirety).

The use of reconstituted bovine collagen to correct defects or augmenttissue also possesses inherent limitations. For example, it has beenreported that reconstituted bovine collage is only moderately effective,and is associated with infrequent, but controversial, adverse reactions.In addition, it is rapidly broken down and resorbed in vivo, providingonly a temporary correction of a skin defect or augmentation. Moreimportantly, reconstituted bovine collagen may elicit an immune responsein the subject. Id.

As explained above, gelatin matrix implant such as that sold under themark “FRIBEL”, is a composite material of porcine gelatin powder ando-aminocaproic acid which are dispersed in 0.9% (by volume) sodiumchloride solution and an aliquot of the recipient's plasma mixed in a1:1 ratio, is also used to correct skin defects and augment tissue.However, this material also possess inherent limitations. Specificallygelatin matrix does not appear to have applications in the treatment ofwrinkle lines. Moreover, since a large bore needle (27 gauge or greater)is used to inject the gelatin matrix into the subject's skin, treatmentwith gelatin results in greater discomfort and pain to the subject asopposed to the injection of other fillers. Id.

In addition, the use of autologous fat injections to correct a skindefect or augment tissue in a subject, while eliminating the potentialof eliciting an immune response, also possesses disadvantages. Morespecifically, prior to its injection, fat must be processed by skilledclinicians in aseptic conditions to maintain sterility. In addition, theinjections are not dermal but are subcutaneous or subdermal. Also, avery large bore needle (as large as 16 gauge) is needed to inject thefat into a subject, resulting in great pain, moderate bruising, andformation of visible puncture holes. Moreover, fat injections aresubject to rapid resorption, and must be repeated in order to maintainskin augmentation or defect correction.

The use of autologous, injectable dermal collagen to correct defects oraugment tissue has also met with limited success. For example, if largeconcentrations of collagen are injected, a 27 gauge needle or larger isused, resulting in the infliction of pain on the subject. Furthermore,serial injections are required in order to compensate for the gradualresorption of autologous collagen.

Hence, what is needed is an efficient non-surgical composition thatpromotes the regeneration of tissues of the gums or the palate and bonethat have degenerated as a result of periodontal disease or trauma.

Moreover, what is needed is a composition and method for promotingregeneration of tissue that does not elicit an immune response in thesubject at the site of desired tissue regeneration.

What is also needed is a composition that can be used in non-surgicalmethods to correct defects in skin, such as scars or wrinkles, andaugment tissue in a subject, particularly facial tissue, which is notrapidly resorbed by the body so that additional injections are required.

U.S. Pat. No. 5,591,444, U.S. Pat. No. 5,660,850, and U.S. Pat. No.5,665,372 are incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention provides a composition for promoting regenerationof tissue in a subject that has degenerated as a result of a disease ordisorder, and a method of using the composition that does not sufferfrom the shortcomings of other methods described above. The presentinvention is based on the inventors' discovery of the successful use ofautologous fibroblasts, with and without various forms of matrix, filleror carrier material, to regenerate tissue in a subject, correct skindefects in the subject, or augment tissue in the subject. Fibroblastsare connective-tissue cells involved in tissue repair. When a tissue isinjured, nearby fibroblasts migrate into the wound, proliferate, andproduce large amounts of collagenous matrix, which helps to isolate andrepair the damaged tissue. [Alberts et al., Molecular Biology of theCell, p. 987, 2nd ed., (1992)].

Broadly, the present invention extends to a method for regenerating asubject's tissue (a) that has degenerated as a result of a disease ordisorder or (b) that has a defect, comprising the steps of providing apharmaceutical composition comprising autologous, passaged fibroblasts,identifying a site of tissue degeneration, and injecting an effectiveamount of the composition into tissue at the site of tissue degenerationso that the tissue is augmented and regeneration of tissue is promoted.

Injection of a pharmaceutical composition of the present invention canbe into tissues of the subject comprising the periodontal pocket and/orthe periodontal tissue adjacent to the area of degeneration or intotissue subadjacent to a defect in the oral mucosa, or into the tissue ofthe palate of a human subject, in order promote regeneration of tissuein the oral mucosa, the gingiva, or the palate. Typical defects in theoral mucosa or palate that can be corrected with this embodiment of thepresent invention include those caused by trauma, dermatoses, recurrentaphthous stomatitis, and infections, or a disease or disorder. Moreover,the present invention can be used to correct defects in the skin, suchas scars, wrinkles, laugh lines, rhytids, stretch marks, depressedscars, cutaneous depressions of non-traumatic origin, acne scarring, orsubcutaneous atrophy from acne, trauma, congenital malformation, oraging. Moreover, the invention can be used to treat defects such ahypoplasia of the lips, or labial folds. In addition, the compositioncan be used to repair a defect, disorder or disease of bone, e.g., bonessuch as, for example, facial bones including orbits, mandibles,maxillae, zygomatic bones, crania, and nasal bones. Bone diseases,disorders, or defects, include, for example, tooth extraction-relatedbone defects or those due to periodontal disease.

A disease or disorder which results in tissue degeneration in a subjectthat can be treated with the present invention includes, but is notlimited to, defects of the oral mucosa, periodontal disease, trauma tothe oral mucosa (e.g., extraction of a tooth), diabetes, cutaneousulcers, or venous stasis. Moreover, periodontal disease can includeperiodontal degeneration, gingivitis, or a non-healing wound of thepalatal mucosa or the gingival mucosa.

The present invention further extends to a method of forming acomposition comprising autologous, passaged fibroblasts which aresubstantially free of immunogenic proteins, such as culture mediumserum-derived proteins, and are histocompatible with a subject. Thismethod comprises the steps of collecting a biopsy of dermis from asubject, isolating the autologous fibroblasts contained in the biopsyfrom extracellular matrix and other cells contained in the biopsy,culturing the autologous fibroblasts in a culture medium that permitsexpansion of the autologous fibroblasts, incubating the autologousfibroblasts in a protein free medium for at least about 2 hours betweenabout 30° C. and about 37.5° C., and exposing the incubated autologousfibroblasts to a proteolytic enzyme so as to suspend the fibroblasts. Anexample of a culture medium that permits expansion of autologousfibroblasts comprises between 0.0% and about 20% serum, wherein theserum can be either human or non-human. Also, the biopsy from dermis cancomprise tissue from the gums, palate or skin of the subject. Henceautologous fibroblasts from the gums, plate or skin have applications inthe present invention.

In another embodiment, the passaged autologous fibroblasts can be addedto a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition. In producing a pharmaceutical composition of the presentinvention, immunogenic proteins, e.g., culture medium serum-derivedproteins, are removed from the autologous fibroblasts, thereby avoidingan immunological reaction in a subject when such cells are reintroducedto the subject proximate to the site of tissue degeneration or defect.

In addition, the present invention extends to a device for delivering apharmaceutical composition of the present invention to a site proximateto the site of tissue degeneration or defect in a subject, wherein thedevice comprises a hypodermic syringe having a syringe chamber, a pistondisposed therein, an orifice communicating with the chamber, apharmaceutical composition comprising autologous passaged fibroblastsand a pharmaceutically acceptable carrier, such that the pharmaceuticalcomposition is disposed in the chamber, and a hypodermic needle is fixedto the orifice.

Tissues which have suffered degeneration or have a defect that can betreated with a device of the present invention include the oral mucosa,the gingival mucosa, and the palatal mucosa. Moreover, diseases ordisorders which can be treated with this device include periodontaldegeneration, gingivitis, or a non-healing wound of the palatal mucosaor the gingival mucosa. Other defects that can be treated with thisinvention include those listed above.

The present invention further extends to a composition for repairingtissue that has degenerated in a subject as result of a disease,disorder or defect in the subject, wherein the composition comprises abiodegradable acellular matrix, and autologous passaged fibroblasts(derived, for example, from the gums, palate, or skin of the subject)and is substantially free of immunogenic proteins, e.g., culture mediumxenogeneic (e.g., fetal bovine) serum-derived proteins, wherein theautologous fibroblasts are integrated into the biocompatiblebiodegradable acellular matrix. In an embodiment of the presentinvention, the biocompatible biodegradable acellular matrix comprisesexogenous proteins, such as any type of collagen. In addition, thebiodegradable acellular matrix can be comprised of any type of collagenand glycosaminoglycans (GAG) cross-linked with, for example,glutaraldehyde, or any type of collagen.

In yet another example, the biodegradable acellular matrix comprises oneor more of gelatin, polyglycolic acid, cat gut, demineralized bone, orhydroxyapatite. Other appropriate matrices consist of bone from whichsubstantially all (e.g., at least 80%, at least 90%, at least 95%, atleast 99%, or even 100% by weight) organic material has been removed(referred to herein as “anorganic bone”); such matrices can, optionally,include exogenous collagen in various amounts (e.g., about 1%, about 2%,about 5%, about 10%, or about 20% by dry weight).

Also, diseases, disorders or defects resulting in degeneration of tissuein a subject which can be treated with the present invention, comprisedefects of the oral mucosa, trauma (e.g., extraction of a tooth) to theoral mucosa or oral bones such as the maxillary or mandibular bones,periodontal disease, diabetes, cutaneous ulcers, or venous stasis. Inaddition, examples of periodontal disease which result in tissuedegeneration include, but are not limited to, periodontal degeneration,gingivitis, or non-healing wounds of the palatal mucosa or gingivalmucosa, or bone degeneration. Other defects that can be treated withthis invention include skin defects, such as scars, wrinkles, laughlines, stretch marks, depressed scars, cutaneous depressions ofnon-traumatic origin, acne scarring, or subcutaneous atrophy from acne,trauma, congenital malformation, or aging. Moreover, the invention canbe used to treat defects such a hypoplasia of the lips, labial folds, orbone defects, e.g., defects of bones such as, for example, facial bonesincluding orbits, mandibles, maxillae, zygomatic bones, crania, andnasal bones.

Also encompassed by the invention is a method for making a compositionfor the repair of tissue that has degenerated in a subject as result ofa disease, disorder, or defect in the subject. The method comprises:providing a suspension of autologous, passaged fibroblasts; providing abiodegradable acellular matrix; incubating the suspension of autologouspassaged fibroblasts with the biodegradable acellular matrix such thatthe autologous passaged fibroblasts integrate within the biodegradableacellular matrix; and removing substantially all culture mediumserum-derived proteins from said biodegradable acellular matrix and saidintegrated fibroblasts to form a composition for promoting the repair oftissue. Sufficient autologous, passaged fibroblasts integrate within thebiodegradable acellular matrix to substantially fill the space on andwithin the biodegradable acellular matrix available for cells. As usedherein, “substantially” fill with passaged autologous fibroblasts meansto fill to a level sufficient to prevent an amount of cell proliferationthat degrades a collagen matrix to a practically deleterious level, as aperson skilled in the art can readily determine for a particularembodiment.

The biodegradable acellular matrix used in this method can containexogenous protein such as, for example, any type of collagen, e.g., anytype of collagen and glycosaminoglycans, cross-linked with, for example,glutaraldehyde. The biodegradable acellular matrix can contain one ormore of the following substances: gelatin, polyglycolic acid, cat gut,demineralized bone, hydroxyapatite, gelatin, polyglycolic acid, cat gut,or anorganic bone with or without any of range of concentrations ofexogenous collagen (see below).

The disease, disorder, or defect to be treated can be a defect of anoral mucosa, trauma to an oral mucosa, periodontal disease, diabetes, acutaneous ulcer, venous stasis, a scar of skin, or a wrinkle of skin.Alternatively, the disease or disorder can be periodontal disease, andthe periodontal disease can be periodontal degeneration, gingivitis, ora non-healing wound of a palatal mucosa or a gingival mucosa.

In this method the step of providing a suspension of autologous,passaged fibroblasts can involve: collecting a biopsy of dermis orpalate of the subject; separating dermal autologous fibroblasts from thebiopsy; culturing the dermal autologous fibroblasts in a culture mediumcontaining (a) between 0.0% and about 20% human or non-human serum and(b) a reagent that prevents the growth of mycoplasma; and exposing theincubated dermal autologous fibroblasts to a proteolytic enzyme so as tosuspend fibroblasts. The step of collecting a biopsy of dermis caninvolve collecting a biopsy from gums, palate or skin of the subject.The reagent can contain tylosin and, optionally, one or more of thefollowing compounds: gentamicin, ciprofloxacine, alatrofloxacine,azithromycin, or tetracycline.

The present invention further extends to a method of using a compositionfor promoting regeneration of tissue, wherein the method comprisesproviding passaged autologous fibroblasts integrated into abiodegradable acellular matrix, identifying a site (i) of tissuedegeneration due to a disease or disorder in the subject or (ii) adefect in the tissue of the subject, and placing the composition on thesite so that the tissue is repaired. Autologous passaged fibroblastsused herein can comprise fibroblasts from the gums, palate or skin ofthe subject.

Diseases, disorders, or defects which can be treated with this methodinclude, but are not limited to, defects of the oral mucosa, trauma tothe oral mucosa (e.g., extraction of a tooth), periodontal disease,diabetes, cutaneous ulcers, or venous stasis. Examples of periodontaldisease that can be treated with the present invention compriseperiodontal degeneration, gingivitis, or a non-healing wound of thepalatal mucosa or the gingival mucosa. Moreover, defects (e.g., skindefects such as scars or wrinkles) can be treated with the compositionof the present invention. In a preferred embodiment, such defects aretreated with a composition comprising fibroblasts from the palate. Anyof the above listed diseases, disorders, or defects can also be treatedby these methods.

Furthermore, biodegradable acellular matrices having applications in thepresent invention may comprise exogenous proteins. Examples of suchmatrices include matrices comprising any type of collagen, or any typeof collagen and glycosaminoglycans (GAG) cross-linked with, for example,glutaraldehyde.

Other examples of biodegradable acellular matrices having applicationsin the present invention include one or more of gelatin, polyglycolicacid, cat gut, demineralized bone (e.g., demineralized human bone), orhydroxyapatite. Other appropriate matrices consist of bone from whichsubstantially all (e.g., at least 80%, at least 90%, at least 95%, atleast 99%, or even 100% by weight) organic material has been removed(referred to herein as “anorganic bone”); such matrices can, optionally,include exogenous collagen, in various amounts (e.g., about 1%, about2%, about 5%, about 10%, or about 20% by dry weight).

The present invention further extends to an injectable composition forcorrecting a defect in skin of a subject, or augmenting tissue of asubject, said injectable composition comprising passaged, autologousfibroblasts substantially free of immunogenic proteins, e.g., culturemedium serum-derived proteins, and a biodegradable, acellular injectablefiller material. Passaged autologous fibroblasts having applications inan injectable composition of the present invention are from gums, palateor skin of the subject.

Furthermore, the present invention extends to an injectable compositionas described above, wherein the biodegradable, acellular injectablefiller material comprises endogenous proteins. In particular, theacellular injectable filler material of an injectable composition of thepresent invention comprises an injectable dispersion of autologouscollagen fibers having a concentration in the composition of at least 24mg/ml of composition.

In addition, the present invention extends to an injectable compositionas described above, wherein the biodegradable acellular injectablefiller material comprises exogenous proteins, such as any type ofcollagen. An example of an exogenous collagen having applications in aninjectable composition of the present invention is reconstituted bovinecollagen fibers cross-linked with glutaraldehyde.

Furthermore, the filler material can comprise any type of solubilizedgelatin either alone, or in combination with other materials. In aparticular example, the filler material comprises porcine gelatin powderand o-aminocaproic acid dispersed in sodium chloride solution and analiquot of plasma from the subject to be injected with the composition.Preferably the ratio of sodium chloride to serum is 1:1 by volume. Otherexamples of materials having applications in the present invention asbiodegradable, acellular injectable filler material include, but are notlimited to polyglycolic acid or cat gut.

The present invention further extends to a method for correcting adefect in skin of a subject, or augmenting tissue of a subject, whereinthe method comprises injecting an effective amount of an injectablecomposition comprising autologous passaged fibroblasts substantiallyfree of immunogenic proteins (e.g., culture medium serum-derivedproteins) and a biodegradable, acellular injectable filler material,into the skin of the subject at the site of the skin defect or desiredtissue augmentation, so that regeneration of tissue at the site ispromoted at the site.

Moreover, the present invention extends to a method for correcting adefect in skin of a subject, or augmenting tissue of a subject, themethod comprising the steps of injecting autologous fibroblastssubstantially free of immunogenic proteins, e.g., culture mediumserum-derived proteins, into the subject at a site of a skin defect ordesired tissue augmentation, and subsequently injecting a biodegradable,acellular injectable filler material into the site. In a particularembodiment of this method of the present invention, the duration betweeninjecting the autologous fibroblasts into the subject and injecting thebiodegradable acellular injectable filler into the subject is about twoweeks.

Autologous fibroblasts having applications in methods of the presentinvention for correcting a defect in skin of a subject, or augmentingtissue of a subject can be obtained from the gums, palate of skin of thesubject.

The present invention further extends to a method for correcting adefect in skin of a subject, or augmenting tissue of the subject, asdescribed above, wherein the biodegradable, acellular injectable fillermaterial comprises endogenous proteins. For example, the biodegradableacellular injectable filler material can comprise an injectabledispersion of autologous collagen fibers, preferably at a concentrationof at least 24 mg of autologous fibers per ml of composition.

Furthermore, the present invention extends to a method for correcting adefect in skin or other tissues of a subject (such as those describedabove), or augmenting tissue of the subject, also as described above,wherein the biodegradable, acellular filler material of the compositioncomprises exogenous proteins such as, for example, any type of collagen.An example of collagen having applications in a method of the presentinvention comprises reconstituted bovine collagen fibers cross-linkedwith, for example, glutaraldehyde.

Other examples of biodegradable, acellular injectable filler materialfor use in a method for correcting a defect in skin or a subject, oraugmenting tissue of the subject include, but are not limited tosolubilized gelatin, polyglycolic acid, or cat gut sutures. Morespecifically, an example of acellular injectable filler material havingapplications in the present invention comprises porcine gelatin powderand aminocaproic acid dispersed in sodium chloride solution, and analiquot of plasma from the subject. Preferably, the ratio of sodiumchloride solution to the aliquot of serum is 1:1 by volume. Furthermore,the sodium chloride solution comprises 0.9% sodium chloride by volume.

In addition, the present invention extends to a method for correcting adefect in skin of a subject, or augmenting tissue of a subject, asdescribed above, wherein the ratio of autologous fibroblastssubstantially free of immunogenic proteins (e.g., culture mediumserum-derived proteins) biodegradable, acellular injectable fillermaterial is approximately 1:1 by volume.

Accordingly, it is an object of the present invention to provide acomposition for augmenting tissue, or promoting regeneration of tissuesuch as the oral mucosa, the gingival mucosa, or the palatal mucosa orskin, which has degenerated as a result of a disease or disorder.Examples of such disorders include periodontal disease, trauma,dermatoses, recurrent aphthous stomatitis, infections, scars, orwrinkles and the others listed above.

It is another object of the present invention to provide a compositionfor augmenting tissue, or promoting regeneration of tissue, wherein thecomposition is histocompatible with a subject, thereby avoidingelicitation of an immune response and inflammation in the tissues of thesubject near the site of degeneration of tissue.

It is yet another object of the present invention to provide method ofpromoting tissue regeneration that does not require surgery.

It is yet still another object of the present invention to promoteregeneration of tissue in a subject without the use of antibiotics inthe subject, and hence prevent the emergence of antibiotic resistantpathogens and deleterious side effects associated with antibiotics inthe subject.

It is another object to provide an injectable composition for correctingdefects in skin, such as scars or wrinkles, or for augmenting tissue ina subject, particularly facial tissue (such as lips), which includespassaged autologous fibroblasts that can withstand resorption so thatsubsequent injections are not needed, and to prevent the elicitation ofan immune response in the subject.

It is yet another object of the present invention to provide methods forcorrecting defects in skin, such as scars or wrinkles, or for augmentingtissue in a subject, that employs the injectable composition set forthabove, that inflicts limited pain on the subject, and does not elicit animmune response in the subject, and is not rapidly resorbed.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the recognition that livingcells normally present in tissue that has degenerated, particularlyfibroblasts, are the ideal material to augment the volume of tissue inorder to promote regeneration of tissue. Hence the present inventionameliorates and reverses the degenerative effects of a disease ordisorder which results in tissue degeneration.

Additionally, the present invention is based on the recognition that anideal composition with which to augment the oral mucosa subadjacent to adefect, to treat defects in the palate or gingiva, or to promoteregeneration of tissue that has degenerated as a result of periodontaldisease, would comprise living cells normally present in such tissues,particularly fibroblasts.

Moreover, the present invention is based on the recognition that anabundant supply of autologous cells of the desired type can be obtainedby culturing a biopsy specimen taken from the skin, palate or gums of asubject several weeks prior to treating the tissue degenerating disease,disorder, or defect. The invention is further based on the recognitionthat, after such a tissue culture expansion, the autologous cells willcontain a significant quantity of immunogenic proteins, e.g., proteinsderived from xenogeneic (e.g., bovine, horse, goat, or sheep) serum usedto supplement the medium used for tissue culture, but that theimmunogenic proteins can be removed, prior to treatment of the subject.

The term “biodegradable” as used herein denotes a composition that isnot biologically harmful and can be chemically degraded or decomposed bynatural effectors (e.g., weather, soil bacteria, plants, animals).

The term “autologous” as used herein refers to cells removed from adonor and administered to a recipient, wherein the donor and recipientare the same individual.

The term “effective amount” as used herein refers to the injection of anamount of pharmaceutical composition of the present invention to promotetissue regeneration in of tissue that has degenerated in a subject.

As used herein, a composition or cells (e.g., autologous passagedfibroblasts) that are “substantially free of culture mediumserum-derived proteins” are a composition or cells in which the fluidsurrounding the composition or cells or incorporated into the body ofbiodegradable acellular matrices that are components of suchcompositions contains less than 0.1% (e.g., less than 0.02, 0.04, 0.008,or 0.0016%) of the xenogeneic serum contained in the tissue culturemedium in which the composition or cells were last cultured.

EXAMPLE I Administration of a Suspension of Autologous Fibroblasts toPromote Tissue Regeneration and Correct Defects in Tissues Method ofObtaining an Injectable Cell Pharmaceutical Composition

As disclosed above, one embodiment of the present invention comprises amethod for regenerating tissue that has been damaged in a subject as aresult of a disease or disorder in the subject, wherein the methodcomprises providing a pharmaceutical composition comprising ofautologous, passaged fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins), identifying asite of tissue degeneration or a defect in tissue, injecting aneffective amount of the pharmaceutical composition into the tissue atthe site of the tissue degeneration or defect so that the tissue isaugmented, and the growth of tissue is promoted at the site ofdegenerated tissue.

A disease or disorder which promotes tissue degeneration in a subject,and can be treated with this aspect of the present invention includes,but is not limited to, defects of the oral mucosa, trauma to the oralmucosa, periodontal disease, diabetes, cutaneous ulcers, or venousstasis. Furthermore, examples of periodontal disease which can betreated with this aspect of the present invention include periodontaldegeneration, gingivitis, or a non-healing wound of the palatal mucosaor the gingival mucosa. In addition, defects in skin and other tissues(see above) can be treated with the present invention.

The invention can be practiced by injecting any undifferentiatedmesenchymal cell that can be expanded in culture. In a preferredembodiment, dermal fibroblasts are injected because they can be readilyobtained and expanded, and because they are a cell type normally presentbeneath the gingival mucosa or palatal mucosa. Fibroblasts taken from abiopsy of the gums, palate, or skin of the subject can be used in thepresent invention.

In providing a composition of the present invention, a dermal fibroblastculture is initiated from a 1 to 5 mm full thickness biopsy specimen ofthe gums, palate or skin of a subject suffering from tissuedegeneration. Because of the phenomenon of allograft rejection, which iswell known to transplantation surgeons and immunologists, it isessential that the cultured fibroblasts be histocompatible with thehost. Histocompatibility can be ensured by obtaining a biopsy of thesubject to be treated and culturing the fibroblasts from this specimen.

Before the initiation of the culture, the biopsy is washed repeatedlywith antibiotic and antifungal agents. An exemplary “wash medium” cancontain tissue culture medium such as Dulbecco's Modified Eagle's Medium(DMEM) and all or some of the following antibiotics: gentamicin,amphotericin B (Fungizone), and tylosin (manufactured by Gibco-BRL andsold by Life Technologies, Rockville, Md., as “Anti-PPLO”, PPLO being anacronym for “pleuropneumonia-like organism”, now known as “mycoplasma”).Gentamicin can be used at a concentration of 0.1-5.0 (e.g., about 0.5)mg/ml. Amphotericin B can be used at a concentration of 0.0005-0.0125(e.g., about 0.0025) mg/ml. Tylosin can be used at a concentration of0.012-1.2 (e.g., 0.12) mg/ml. The specimen of dermis is then separatedinto small pieces. The pieces of the specimen are individually placedonto the dry surface of a tissue culture flask and allowed to attach forbetween about 5 and about 10 minutes, before a small amount of medium isslowly added, taking care not to displace the attached tissue fragments.After about 48 hours of incubation, the flask is fed with additionalmedium. When a T-25 flask is used to start the culture, the initialamount of medium is about 1.5-2.0 ml. The establishment of a cell linefrom the biopsy specimen ordinarily takes between about 2 and 3 weeks,at which time the cells can be removed from the initial culture vesselfor expansion.

During the early stages of the culture, it is desirable that the tissuefragments remain attached to the culture vessel bottom; fragments thatdetach should be reimplanted into new vessels. The fibroblasts can bestimulated to grow by a brief exposure to EDTA-trypsin, according totechniques well known to those skilled in the art. The exposure totrypsin is too brief to release the fibroblasts from their attachment tothe culture vessel wall. Immediately after the cultures have becomeestablished and are approaching confluence, samples of the fibroblastscan be processed for frozen storage, such as in liquid nitrogen.Presently, numerous methods for successfully freezing cells for lateruse are known in the art, and are included in the present invention. Thefrozen storage of early rather than late passage fibroblasts ispreferred because the number of passages in cell culture of normal humanfibroblasts is limited.

The fibroblasts can be frozen in any freezing medium suitable forpreserving fibroblasts. A medium consisting of about 70% (v/v) growthmedium, about 20% (v/v) fetal bovine serum and about 10% (v/v)dimethylsulfoxide (DMSO) can be used with good effect. DMSO can also besubstituted with, for example, glycerol. Thawed cells can be used toinitiate secondary cultures to obtain suspensions for use in the samesubject without the inconvenience of obtaining a second specimen.

Any tissue culture technique that is suitable for the propagation ofdermal fibroblasts from biopsy specimens may be used to expand the cellsto practice the invention. Techniques well known to those skilled in theart can be found in R. I. Freshney, Ed., ANIMAL CELL CULTURE: APRACTICAL APPROACH (IRL Press, Oxford, England, 1986) and R. I.Freshney, Ed., CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES,Alan R. Liss & Co., New York, 1987), which are hereby incorporated byreference.

The medium can be any medium suited for the growth of primary fibroblastcultures. The medium can be supplemented with human or non-human serumin an amount of between about 0.0% and about 20% (v/v) to promote growthof the fibroblasts. Higher concentrations of serum promote faster growthof the fibroblasts. An example of medium having application hereincomprises glucose DMEM supplemented with about 2 mM glutamine, about 10mg/L sodium pyruvate, about 10% (v/v) fetal bovine serum and antibiotics(“complete medium”), wherein the concentration of glucose ranges fromabout 1,000 milligrams per liter of medium to about 4,500 milligrams perliter of medium. Fibroblasts can also be expanded in serum-free medium.Tissue culture growth medium used for culturing the fibroblasts isgenerally supplemented with antibiotics to prevent microbial (e.g.,bacterial, fungal, yeast, and mycoplasma) contamination of the cultures.Mycoplasma contamination is a frequent and particularly vexatiousproblem in tissue culture. In order to prevent or minimize mycoplasmacontamination, an agent such as tylosin can be added to the relevantculture medium. The medium can be further supplemented with one or more(e.g., all) the following antibiotics: gentamicin, ciprofloxacine,alatrofloxacine, azithromycin, and tetracycline. Tylosin can be used ata concentration of 0.006-0.6 mg/ml (e.g., about 0.06) mg/ml. Gentamicincan be used at a concentration of 0.02-0.5 (e.g., about 0.1) mg/ml.Ciprofloxacine can be used at concentration of 0.002-0.05 (e.g., about0.01) mg/ml. Alatrofloxacine can be used at a concentration of 0.2-5.0(e.g., about 1.0) μg/ml. Azithromycin can be used at a concentration of0.002-0.05 (e.g., 0.01) mg/ml. Tetracycline can be used at aconcentration of 0.004-0.1 (e.g., about 0.02) mg/ml. The antibiotics canbe present for the whole period of the culture or for only part of theculture period.

Mycoplasmal contamination was tested for by an agar culture method usinga culture system such as, for example, a kit marketed by Sigma, St.Louis, Mo., as “Mycoplasma Test Medium” (Cat. No. M 1914) and by PCR.PCR testing was performed by the both the ATCC (Manassas, Va.) andEsoterics Co., Houston, Tex. The ATCC markets a PCR mycoplasma test kit(“Mycoplasma Detection Kit”; cat. no. 90-100K). Using both the agarculture method and the PCR test, a low level of mycoplasmalcontamination was detected in four out of fifteen cultures performed inmedium supplemented with gentamicin as the only antibiotic; on the otherhand, no mycoplasmal contamination was detected in any of 12 culturescontaining tylosin (0.06 mg/ml), gentamicin (0.1 mg/ml), ciprofloxacine(0.01 mg/ml), alatrofloxacine (1.0 μg/ml), azithromycin (0.01 mg/ml),and tetracycline (0.02 mg/ml). The antibiotic mixture was present in thefibroblast cultures only for the first two weeks after initiation. Aftertwo weeks of culture antibiotic containing medium was replaced withantibiotic-free medium. Once sufficient cells had grown, they weretested for mycoplasmal as well as bacterial and fungal contamination.Only cells with no detectable contamination were used in the describedtreatment methods of the invention. Another agent that has been found bythe inventors to be useful in preventing mycoplasmal contamination is aderivative of 4-oxo-quinoline-3-carboxylic acid (OQCA) which is sold asMycoplasma Removal Agent (MRA) by ICN Pharmaceuticals, Inc. (Costa Mesa,Calif.) and was used according to the manufacturer's instructions. Thisderivative of OQCA can be used at a concentration of 0.1-2.5 (e.g., 0.5)μg/ml.

Autologous fibroblasts can be passaged into new flasks bytrypsinization. For expansion, individual flasks are split 1:3. Triplebottom, T-150 flasks, having a total culture area of 450 cm², aresuitable for the practice of the invention. A triple bottom T-150 flaskcan be seeded with about 1×10⁶ to about 3×10⁶ cells and has a capacityto yield about 8×10⁶ to about 1.0×10⁷ cells. When the capacity of theflask is reached, which typically requires about 5-7 days of culture,the growth medium is replaced by serum-free medium; thereafter the cellsare incubated, i.e., held at between bout 30° C. and about 37.5° C., forat least 4 hours (e.g., overnight or about 18 hours). The incubation ofthe cells in serum free medium substantially removes from the cellsproteins that are derived from the fetal bovine serum which, if present,can elicit an untoward immune response in the subject. In a preferredembodiment, serum-free medium comprises glucose DMEM supplemented withabout 2 mM glutamine, and about 110 mg/L sodium pyruvate, wherein theconcentration of glucose can range from approximately 1,000 mg/L ofmedium to about 4,500 mg/L of medium. In a preferred embodiment, theconcentration of glucose is approximately 4,500 mg/L of medium. Theserum-free medium can also contain the above-described antibiotics.

At the end of the incubation in serum free medium, the cells are removedfrom the tissue culture flask by trypsin-EDTA; washed extensively bycentrifugation and resuspension; and suspended for injection in an equalvolume of injectable isotonic solution with an appropriate physiologicalosmolarity, which is substantially pyrogen and foreign protein free. Anexample of such an isotonic solution is isotonic saline. Five triplebottom T-150 flasks, grown to capacity, yield about 3.5×10⁷ to about7×10⁷ cells which are sufficient to make up about 1.2 to about 1.4 ml ofsuspension. A pharmaceutically acceptable carrier can then be added tothe passaged autologous fibroblasts forming a pharmaceuticalcomposition. The phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are not deleterious to thecells, are physiologically tolerable, and do not typically produce anallergic or similar untoward reaction, such as gastric upset, dizzinessand the like, when administered to a human. Such compositions includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength.

Alternatively, the cells can be transported on ice at about 4° C. solong as they are injected within 24-48 hours of the time that thepharmaceutical composition is made. The cells can be suspended in anappropriate physiological solution with appropriate osmolarity andtested for pyrogen and endotoxin levels, except for the absence ofphenol red pH indicator, and the replacement of the fetal bovine serumby the subject's serum for such transportation (transport medium). Inanother embodiment the cells can be suspended in Krebs-Ringer solutioncomprising 5% dextrose or any other physiological solution. The cellscan be aspirated and injected in the transport medium.

The volume of saline or transport medium in which the cells aresuspended is related to such factors as the number of fibroblasts thepractitioner desires to inject, the extent of the damage due to tissuedegeneration or defect, or the size, the number of the defects that areto be treated, and the urgency of the subject's desire to obtain theresults of treatment. Moreover, the practitioner can suspend the cellsin a larger volume of medium and inject correspondingly fewer cells ateach injection site.

Device for Treating Degenerated Tissue with Autologous PassagedFibroblast Pharmaceutical Composition of the Present Invention

As explained above, the present invention extends to a device deliveringa pharmaceutical composition of autologous passaged cells describedabove, to a point proximate to the site of tissue degeneration, ordefects of the palate, oral mucosa or skin. Such a device comprises ahypodermic syringe having a syringe chamber, a piston disposed therein,an orifice communicating with the chamber, a pharmaceutical compositioncomprising autologous, passaged fibroblasts, and a pharmaceuticallyacceptable carrier thereof, such that the pharmaceutical composition isdisposed in the chamber, and a hypodermic needle is fixed to theorifice.

The Administration of a Pharmaceutical Composition

A pharmaceutical composition of the invention can be used to treattissue degeneration in a subject as a result of a disorder or disease,such as periodontal disease, or defects of the oral mucosa or skin, suchas scars or wrinkles, by use of the following techniques.

Initially, the tissue to be injected is prepped with alcohol andstretched to give a taut surface. If the tissue degeneration is theresult of periodontal disease, the tissue to be injected is periodontaltissue, including periodontal pockets. If the tissue to be injectedcontains defects in the palate or gums of the subject, the tissue to beinjected with a pharmaceutical composition of the present invention issubadjacent to the defect. Also, if the tissue to be injected is theskin, in order to treat defects, e.g., scars, wrinkles, or any of thedefects listed above, it is also injected into the dermis orsubcutaneous tissue.

After the tissue to be injected has been prepped, a syringe is filledwith a pharmaceutical composition of the present invention and fittedwith a 30 gauge needle. The needle is inserted into the tissue assuperficially as possible, and the orientation of the bevel is notcritical to the success of this method of the present invention. Theinjection of the pharmaceutical composition is made by gentle pressureuntil a slight blanch is seen in the injected tissue. Multiple serialinjections are made.

EXAMPLE II A Composition for Promoting the Regeneration of Tissue thathas Degenerated in a Subject

Also disclosed in the present invention is a composition for promotingthe regeneration of tissue that degenerated in a subject. Suchdegeneration can occur as a result of periodontal disease, trauma,dermatoses, recurrent aphthous stomatitis, or infections to name only afew. Other relevant diseases and disorders are listed above. Moreover,examples of periodontal disease which can cause tissue degenerationinclude periodontal degeneration, gingivitis, or non-healing wounds ofthe palatal mucosa or gingival mucosa. Moreover, a composition of thepresent invention can also be used to correct defects in tissue of asubject, such as, for example, defects in the palatal mucosa, gingivalmucosa, or defects in skin, e.g., any of those described above. Suchcompositions can be used, for example, for healing extraction socketsafter extraction of a tooth. Moreover, they can be used to fillextraction-associated bone defects and to rebuild bone in periodontaldisease. They can be used, for example, to repair dental ridges andmandibular bone, maxillary bone, and sinus floor defects. Indeed theycan be used to repair any bony defect in the body, including, forexample, non-union of fractured bones such as long bones. A compositionof the present invention comprises a biodegradable acellular matrix, andautologous passaged fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins), wherein theautologous fibroblasts are integrated into the biodegradable acellularmatrix.

Moreover, disclosed herein is a method of making a composition of thepresent invention to promote regeneration of tissue in a subject. Such amethod comprises providing a suspension of passaged autologousfibroblasts substantially free of immunogenic proteins, e.g., culturemedium serum-derived proteins, providing a biodegradable acellularmatrix, incubating the biodegradable acellular matrix with thesuspension of passaged autologous fibroblasts such that the autologousfibroblasts integrate within the biodegradable acellular matrix forminga composition for promoting regeneration of tissue. Also disclosedherein is a method of using a composition of the present invention,comprising identifying a site of tissue degeneration, and applying thecomposition for promoting regeneration of tissue to the site of tissuedegeneration.

In order to produce a composition of the present invention, autologouspassaged fibroblasts substantially free of immunogenic proteins (e.g.,culture medium serum-derived proteins) must be made available to avoidthe phenomenon of allograft rejection, which is well known totransplantation surgeons and immunologists. Hence, it is essential thatthe cultured fibroblasts be histocompatible with the host.Histocompatibility can be ensured by obtaining a biopsy of the gums,palate or skin of the subject to be treated, and culturing thefibroblasts from this specimen.

Before the initiation of the dermal fibroblast culture, a biopsy of 1-3mm is taken from the gums, palate or skin of the subject, and washedrepeatedly with antibiotic and antifungal agents (see above). Thespecimen of dermis is then separated into small pieces. The pieces ofthe specimen are individually placed onto a dry surface of a tissueculture flask and allowed to attach for between 5 and 10 minutes beforea small amount of medium is slowly added, taking care not to displacethe attached tissue fragments. After 48 hours of incubation, the flaskis fed with additional medium. When a T-25 flask is used to start theculture, the initial amount of medium is 1.5-2.0 ml. The establishmentof a cell line from the biopsy specimen ordinarily takes between 2 and 3weeks, at which time the cells can be removed from the initial culturevessel for expansion.

During the early stages of the culture it is desirable that the tissuefragments remain attached to the culture vessel bottom; fragments thatdetach should be reimplanted into new vessels. The autologousfibroblasts can be stimulated to grow by a brief exposure totrypsin-EDTA, according to techniques known to those skilled in the art.The exposure to trypsin is too brief to release the fibroblasts formtheir attachment to the culture vessel wall.

Immediately after the cultures have become established and areapproaching confluence, samples of the autologous fibroblasts can beprocessed for frozen storage in, for example, liquid nitrogen. Thefrozen storage of early rather than late passage fibroblasts ispreferred because the number of passages in cell culture of normal humanfibroblasts is limited.

The autologous fibroblasts can be frozen in any freezing medium suitablefor preserving cells. A medium consisting of about 70% growth medium,about 20% (v/v) fetal bovine serum and about 10% (v/v) dimethylsulfoxide(DMSO) can be used with good effect. DMSO can also be substituted with,for example, glycerol. Thawed cells can be used to initiate secondarycultures to obtain suspensions for use in the same subject without theinconvenience of obtaining a second specimen.

Any tissue culture technique that is suitable for the propagation ofdermal fibroblasts from biopsy specimens may be used to expand the cellsto practice the invention. Techniques for propagation known to thoseskilled in the art can be found in R. I. Freshney, Ed., ANIMAL CELLCULTURE: A PRACTICAL APPROACH (IRL Press, Oxford England, 1986) and R.I. Freshney, Ed., CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES,Alan R. Liss & Co., New York, 1987), which are hereby incorporated byreference.

The medium can be any medium suited for the growth of primary autologousfibroblast cultures. In most instances, the medium is supplemented withhuman or non-human serum in the amount of between about 0.0% and about20% (v/v) to promote growth of the autologous fibroblasts. Higherconcentrations of serum promote faster growth of the fibroblasts. In apreferred embodiment the serum is fetal bovine serum, which is added toa final concentration of about 10% of medium. Moreover, the medium canbe glucose DMEM supplemented with about 2 mM glutamine, about 110 mg/Lsodium pyruvate, about 10% (v/v) fetal bovine serum and antibiotics(“complete medium”), wherein the concentration of glucose ranges fromapproximately 1,000 mg/L of medium to approximately 4,500 mg/L ofmedium. Preferably, the concentration of glucose in the medium isapproximately 4,500 mg/L of medium. The medium will generally alsocontain antibiotics such those described above.

Autologous fibroblasts can be passaged into new flasks bytrypsinization. For expansion, individual flasks are split 1:3. Triplebottom, T-150 flasks, having a total culture area of 450 cm², aresuitable for the practice of the invention. A triple bottom T-150 flaskcan be seeded with about 1×10⁶ to about 3×10⁶ to about 3×10⁶ cells andhas a capacity to yield about 8×10⁶ to about 1.0×10⁷ cells. When thecapacity of the flask is reached, which typically requires 5-7 days ofculture, the growth medium is replaced by serum-free medium; thereafterthe cells are incubated, i.e., held at between about 30° C. and about37.5° C., for at least 4 hours (e.g., overnight, or about 18 hours). Theincubation of the cells in serum free medium substantially removes fromthe cells proteins that are derived from the fetal bovine serum which,if present, would elicit an immune subject. In a preferred embodiment,the serum-free medium comprises glucose and DMEM supplemented with about2 mM glutamine, about 110 mg/L sodium pyruvate, wherein theconcentration of glucose ranges from approximately 1,000 mg/L of mediumto approximately 4,500 mg/L of medium, and preferably is 4,500 mg/L ofmedium.

A biodegradable acellular matrix is then provided. Examples of suchmatrices which can be used in the present invention include, but are notlimited to, acellular matrices comprising exogenous proteins, ormatrices comprising biodegradable polymers.

Numerous biodegradable acellular matrices comprising exogenous proteinsare presently available, and have ready applications in the presentinvention. An embodiment of such biodegradable acellular matrices arematrices comprising any type of collagen, or any type of collagen withglycosaminoglycans (GAG) cross-linked with, for example, glutaraldehyde.Examples of collagen matrices having application in the presentinvention are absorbable collagen sponges made by the Calcitek Companyof Carlsbad, Calif. These collagen sponge dressings, sold under thenames “COLLATAPE®,” “COLLACOTE®,” and “COLLAPLUG®” are made fromcross-linked collagen extracted from bovine deep flexor (Achilles)tendon, and glycosaminoglycans (GAGS). These products are soft, pliable,nonfriable, and non-pyrogenic. In addition, more than 90% of thisproduct consists of open pores. An alternative biodegradable acellularmatrix can consist of collagen (e.g., bovine porcine collagen type I)formed into a thin membrane. Such a membrane is manufactured by theCalcitek Company and is marketed as BioMend™. Another such membranousmatrix manufactured by ED. GEISTLICH SÖHNE AG of Wolhusen, Switzerland,is made of porcine type I and type III collagen and is marketed asBio-Gide®. Bio-Gide® has a bilayer structure with one surface that isporous allowing the ingrowth of cells and a second surface that is denseand will prevent the ingrowth of fibrous tissue. Another biodegradableacellular matrix can be made from bone spongiosa formed into granules orblocks. This material consists of animal (e.g., human, non-humanprimate, bovine, sheep, pig, or goat) bone from which substantially allorganic material (e.g., proteins, lipids, nucleic acids, carbohydrates,and small organic molecules such as vitamins and non-protein hormones)have been removed. This type of matrix is referred to herein as ananorganic matrix. One such matrix, which is marketed as either Bio-Oss®spongiosa granules or Bio-Oss® blocks, is manufactured by ED. GEISTLICHSÖHNE AG. This company also manufactures a block-type matrix (Bio-Oss®collagen), also consisting of the anorganic bone, but containing inaddition approximately 10% by weight of collagen fibers. Otherbiodegradable acellular matrices having applications in the presentinvention can contain one or more of gelatin, polyglycolic acid, or catgut sutures, demineralized bone, hydroxyapatite, or mixtures of thesesubstances. For example, a matrix made from demineralized human bone andformed into small blocks is marketed as Dynagraft™ matrix by GenSciRegeneration Laboratories, Inc. Demineralized bone can be combined, forexample, with collagen to produce a matrix in the form of a sponge,block, or membrane. Synthetic polymers made from one or more monomerscan also be used to make the biodegradable acellular matrices of theinvention. The matrices can be made from one or more of such syntheticpolymers. The synthetic polymers can also be combined with any of theabove-mentioned substances to form matrices. Different polymers forminga single matrix can be in separate compartments or layers. For example,Gore-Tex, Inc. manufactures a porous biodegradable acellular matrix(GORE RESOLUT XT Regenerative Material) that is composed of a syntheticbioabsorbable glycolide and trimethylene carbonate copolymer fiber (intowhich cells can migrate) attached to an occlusive membrane that does notpermit ingrowth of cells and composed of a synthetic bioabsorbableglycolide and lactide copolymer.

After a biodegradable acellular matrix has been selected, a concentratedsuspension of autologous passaged fibroblasts is evenly distributed onthe surface of the matrix. Using a concentrated suspension is necessaryto avoid going beyond the capacity of the matrix to absorb the liquidsuspension. For example, a typical distribution of cell suspension usingthe GORE RESOLUT XT as the matrix comprises applying about 94 μl toabout 125 μl of cell suspension having about 2.0×10⁶ cells to about4.0×10⁶ cells, per square centimeter of matrix. Cells are allowed toattach to the matrix without further addition of media. In a preferredembodiment, incubation of the cells with the matrix occurs at about 37°C. for about 1-2 hours. After at least sixty minutes of incubation, thecells attach to the matrix material. Histological analysis of suchmatrices after seeding and incubating for at least on hour showed evendistribution of cells throughout the matrices. At this time, the culturevessels containing the cell-loaded matrices are supplemented withadditional growth medium. Cells are then cultured in the matrix forabout 3 to 4 days. The cells are added to the matrix at high density(see above) so as to substantially fill the space within the matrixavailable for cells. As a result, little to no cell proliferation occursduring this 3-4 day culture period. Methods to establish the appropriatenumber of autologous passaged fibroblasts to add to any given acellularbiodegradable matrix would be known to those in the art. Indeed, it isundesirable for significant cell proliferation to occur during thisperiod because the dividing fibroblasts secrete enzymes (e.g.,collagenase) that can degrade or, at least, partially degrade thematrices. The matrix with the cells is then washed at least three times,for 10 minutes per wash, with, e.g., phosphate buffered solution (PBS)to substantially remove immunogenic proteins, e.g., culture mediumserum-derived proteins, which can elicit an immune response in thesubject. Fresh PBS is used for each washing. The matrix is thenincubated twice for at least one hour per incubation in fresh PBS priorto use. After incubation, the matrix comprising autologous fibroblastsis placed on the area of tissue degeneration, or defect, such as, forexample, the periodontal pockets, and secured so that it can not bemoved from the site.

In the case of sponge matrix (e.g. Collacote®), approximately1.5-2.0×10⁷ fibroblasts in approximately 1.5 ml of growth medium areseeded onto 2 cm×4 cm thin (approximately 2.5 mm to 3.0 mm thick)sponges. The sponge is then incubated at 37° C. for about 1-2 hourswithout further addition of medium to allow substantially all thefibroblasts to adhere to the matrix material. After cell adherence,additional growth medium is added to the composition of matrix andfibroblasts which is then incubated at 37° C. for 3-4 days (with dailychange of medium). As explained above, little to no cell proliferationoccurs during this 3-4 day culture. The composition is then removed fromgrowth medium containing FBS and washed repeatedly (at least 3 times)with FBS-free PBS. After each addition of PBS, the matrix is incubatedfor 10-20 minutes prior to discarding of the PBS. After the final wash,the composition is either applied directly to the area of the subjectrequiring tissue regeneration or is transferred to a shipping vialcontaining a physiological solution (e.g., Kreb's Ringer solution) andshipped (preferably overnight) to a practitioner (e.g., a dentist orphysician).

In the case of a membranous matrix (e.g. BioMend™), approximately3-8×10⁶ fibroblasts in approximately 100 μl of growth medium are seededonto the 15 mm×20 mm thin (approximately 0.5 to 1.0 mm thick) membranes.The membrane is then incubated at 37° C. for about 30-60 minutes withoutfurther addition of medium to allow substantially all the fibroblasts toadhere to the matrix material. After cell adherence, additional growthmedium is added to the composition of matrix and fibroblasts which isthen incubated at 37° C. for 2-3 days (with daily change of medium). Thecells were added to the matrix at high density (see above) so as tosubstantially fill the space within the matrix available for cells withthe same result described above. Washing of the composition and eitherimmediate use or shipping are as described above for the spongematrices.

In the case of a block matrix such as the above described anorganicmatrix (e.g., the Bio-Oss® block) or a demineralized bone matrix (e.g.,the Dynagraft™ matrix), approximately 1.2-2.0×10⁷ fibroblasts inapproximately 100 μl to 150 μl of growth medium are seeded into 1 cm×1cm×2 cm cubic blocks of matrix material. Cells are slowly seeded ontoone face of the block face. Once the medium and cells have been absorbedinto the block, another face of the block is seeded in a similarfashion. The procedure is repeated until all faces of the block havebeen seeded and the block is fully saturated with medium. Care is takento avoid adding excess medium and thereby causing leaking out of mediumand cells from the block. The composition is then incubated at 37° C.for about 60-120 minutes without further addition of medium to allowsubstantially all the fibroblasts to adhere to the matrix material.After cell adherence, additional growth medium is added to thecomposition of matrix and fibroblasts which is then incubated at 37° C.for 2-3 days (with daily change of medium). The cells were added to thematrix at high density (see above) so as to substantially fill the spacewithin the matrix available for cells with the same result describedabove. Washing of the composition and either immediate use or shippingare as described above for the sponge matrices.

Compositions using Bio-Oss® collagen, RESOLUT, COLLACOTE®, Dynagraft™ asthe matrix material have been used to heal extraction sockets of 19patients after extraction of a tooth. In untreated extraction sockets,the subject's fibroblasts migrate into the sockets approximately 10-14days after extraction of the tooth and thus the healing process onlybegins at that time. However, by implanting the compositions containingthe patient's fibroblasts into the sockets immediately after extractionof the tooth (e.g., within 1-3 hours of the extraction), the healingprocess is initiated immediately. Furthermore, by implanting thecompositions, shrinkage of the socket (due to collapse of the socketwalls), which is generally approximately 30% in untreated sockets, isminimized. It is particularly desirable that socket shrinkage beminimized in cases in which it is proposed to implant a dentalprosthesis (e.g., a false tooth) at the site of the extraction at alater date.

The inventors have also found that Bio-Oss® collagen matrices loadedwith autologous fibroblasts to be useful in the repair ofextraction-associated mandibular and maxillary dental ridge defects. Intwo patients whose dental ridges had been treated with suchcompositions, after being in place for 6-18 months, there was nodetectable deterioration of the matrix material and the structureresembled normal bone.

In all the compositions used for the above described procedures,fibroblasts obtained from 1 mm punch biopsies of the patients' gumtissues were used and the compositions were shipped to the dentistsperforming the procedures. The compositions were shaped by the dentistto fit into the extraction sockets. In general, the compositions aftershaping had an approximately cylindrical shape with a length ofapproximately 10 mm and a diameter of approximately 4 mm. At the time ofimplanting, fragments of the compositions were prepared for histologicalanalysis. In all cases, fibroblast colonization of and proliferationwithin the matrices was seen by light microscopy.

The above-mentioned advantages (i.e., facile healing and maintenance ofsocket volume) were seen in all cases. Furthermore, infection of thetissues surrounding the socket, which is a frequently observed sequelaof procedures in which extraction sockets are not treated, was notobserved in any case. Compositions using Bio-Oss® collagen (2 patients)as the matrix had the advantage of persistence of the matrix material inthe sockets longer than those compositions in which the matrix materialconsisted of collagen only and thus provided physical support for thefibroblasts before full replacement of the matrix material withcomponents endogenous to the patient, e.g., cells and extracellularmatrix components.

EXAMPLE III An Injectable Composition for Correcting Skin Defects andAugmenting Tissue in a Subject

As explained above, the present invention extends to an injectablecomposition for correcting a defect in skin of a subject, or augmentingtissue of a subject, wherein the injectable composition comprisespassaged autologous fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins), and abiodegradable, acellular injectable filler material. Examples of skindefects that can be treated with the present invention include scars,particularly facial scars resulting from trauma or acne, or wrinkles.Any of the other skin and other tissue defects listed above can also betreated with the present invention. Furthermore, the present inventioncan be used to augment tissue in a subject. For example, the injectablecomposition of the present invention can be injected into the lips of asubject in order to make the lips larger and fuller, or it can beinjected below laugh lines to eliminate or diminish them. Furthermore,the compositions can be injected subcutaneously to treat subcutaneousdefects that can have arisen from congenital or acquired effects.Fibroblasts are used in the present invention because they can bereadily obtained and expanded, and are a cell type normally presentbeneath the dermis. Fibroblasts taken from a biopsy of the gums, palate,or skin of the subject can be used in the present invention, and canreadily obtained using procedures set forth above.

Initially, a dermal fibroblast culture is initiated from a 1 to 5 mmfull thickness biopsy specimen of the gums, palate or skin of thesubject. Because of the phenomenon of allograft rejection, which is wellknown to transplantation surgeons and immunologists, it is essentialthat the cultured fibroblasts be histocompatible with the host.Histocompatibility can be ensured by obtaining a biopsy of the subjectto be treated and culturing the fibroblasts from this specimen.

Before the initiation of the culture, the biopsy is washed repeatedlywith antibiotic and antifungal agents (see above). The specimen ofdermis is then separated into small pieces. The pieces of the specimenare individually placed onto the dry surface of a tissue culture flaskand allowed to attach for between about 5 and about 10 minutes, before asmall amount of medium is slowly added, taking care not to displace theattached tissue fragments. After about 48 hours of incubation, the flaskis fed with additional medium. When a T-25 flask is used to start theculture, the initial amount of medium is about 1.5-2.0 ml. Theestablishment of a cell line from the biopsy specimen ordinarily takesbetween about 2 and 3 weeks, at which time the cells can be removed fromthe initial culture vessel for expansion.

During the early stages of the culture, it is desirable that the tissuefragments remain attached to the culture vessel bottom; fragments thatdetach should be reimplanted into new vessels. The fibroblasts can bestimulated to grow by a brief exposure to EDTA-trypsin, according totechniques well known to those skilled in the art. The exposure totrypsin is too brief to release the fibroblasts from their attachment tothe culture vessel wall.

Immediately after the cultures have become established and areapproaching confluence, samples of the fibroblasts can be processed forfrozen storage, such as in liquid nitrogen. Presently, numerous methodsfor successfully freezing cells for later use are known in the art andare included in the present invention. The frozen storage of earlyrather than late passage fibroblasts is preferred because the number ofpassages in cell culture of normal human fibroblasts is limited.

The fibroblasts can be frozen in any freezing medium suitable forpreserving fibroblasts. A medium consisting of about 70% (v/v) growthmedium, about 20% (v/v) fetal bovine serum and about 10% (v/v)dimethylsulfoxide (DMSO) can be used with good effect. DMSO can also besubstituted with, for example, glycerol. Thawed cells can be used toinitiate secondary cultures to obtain suspensions for use in the samesubject without the inconvenience of obtaining a second specimen.

Any tissue culture technique that is suitable for the propagation ofdermal fibroblasts from biopsy specimens may be used to expand the cellsto practice the invention. Techniques well known to those skilled in theart can be found in R. I. Freshney, Ed., ANIMAL CELL CULTURE: APRACTICAL APPROACH (IRL Press, Oxford, England, 1986) and R. I.Freshney, Ed., CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES,Alan R. Liss & Co., New York, 1987), which are hereby incorporated byreference.

The medium can be any medium suited for the growth of primary fibroblastcultures. The medium can be supplemented with human or non-human serumin an amount of between about 0.0% and about 20% (v/v) to promote growthof the fibroblasts. Higher concentrations of serum promote faster growthof the fibroblasts. An example of medium having application hereincomprises glucose DMEM supplemented with about 2 mM glutamine, about 110mg/L sodium pyruvate, about 10% (v/v) fetal bovine serum and antibiotics(“complete medium”), wherein the concentration of glucose ranges fromapproximately 1,000 mg/L of medium to 4,500 mg/L of medium, andpreferably is 4,500 mg/L. Fibroblasts can also be expanded in serum-freemedium (see above). Serum-containing and serum-free media will generallyalso contain one or more antibiotics such as those listed above.

Autologous fibroblasts can be passaged into new flasks bytrypsinization. For expansion, individual flasks are split 1:3. Triplebottom, T-150 flasks, having a total culture area of 450 cm² aresuitable for the practice of the invention. A triple bottom T-150 flaskcan be seeded with about 1×10⁶ to about 3×10⁶ cells and has a capacityto yield about 8×10⁶ to about 1.0×10⁷ cells. When the capacity of theflask is reached, which typically requires about 5-7 days of culture,the growth medium is replaced by serum-free medium; thereafter the cellsare incubated, i.e., held at between about 30° C. and about 37.5° C.,for at least 2 hours. The incubation of the cells in serum-free mediumsubstantially removes from the cells proteins that are derived from thexenogeneic (e.g., fetal bovine) serum which, if present, can elicit animmune response in the subject. In a preferred embodiment, serum-freemedium comprises glucose DMEM supplemented with about 2 mM glutamine,and about 110 mg/L sodium pyruvate, wherein the concentration of glucoseis approximately 1,000 mg/L of medium to approximately 4,500 mg/L ofmedium, and preferably 4,500 mg/L of medium.

At the end of the incubation in serum-free medium, the cells are removedfrom the tissue culture flask by trypsin-EDTA; washed extensively bycentrifugation and resuspension; and suspended for subsequent use in aninjectable composition of the present invention, or for injection intothe subject. Five triple bottom T-150 flasks, grown to capacity, yieldabout 8×10⁶ to about 1.0×10⁷ cells per flask which are sufficient tomake up about 1.2 to 1.4 ml of cell suspension.

The cells can be transported at on ice 4° C. so long as they areinjected within 24-48 hours of their suspension. The cells can besuspended in an appropriate physiological solution with appropriateosmolarity and tested for pyrogens and endotoxin levels, except for theabsence of phenol red pH indicator, and the replacement of the fetalbovine serum by the subject's serum for such transportation (transportmedium). In another embodiment the cells can be suspended inKrebs-Ringer solution comprising 5% dextrose or any other physiologicalsolution. The cells can be aspirated and injected into the transportmedium.

The volume of saline or transport medium in which the cells aresuspended is related to such factors as the number of fibroblasts thepractitioner desires to inject, the extent of the defects to thesubject's skin that are to be corrected, the size or number of thedefects that are to be corrected, and the urgency of the subject'sdesire to obtain the results of the treatment. Moreover, thepractitioner can suspend the cells in a larger volume of medium andinject correspondingly fewer cells at each injection site. In aninjectable composition of the present invention, the passaged autologousfibroblasts of the present invention are mixed with a biodegradable,acellular injectable filler material in a ratio of approximately 1:1 byvolume.

Biodegradable, Acellular Injectable Filler Material

As explained above, an injectable composition of the present inventionalso comprises a biodegradable, acellular injectable filler material.Numerous types of biodegradable, acellular injectable filler materialsare presently available and have applications in the present invention.More specifically, the filler material can be comprised endogenousproteins, such as any type of collagen from the subject. An example ofsuch a filler is “AUTOLOGEN” produced by Collagenesis, Inc. “AUTOLOGEN”is a dispersion of autologous dermal collagen fibers from the subject,and should not elicit an immune response. In order to obtain “AUTOLOGEN”for the subject, a specimen of tissue is obtained from the subject andforwarded to Collagenesis, Inc., where it is turned into “AUTOLOGEN”.Approximately a one and a half square inch of tissue yields one cubiccentimeter (cc) of “AUTOLOGEN”. After “AUTOLOGEN” has been prepared, itsconcentration can be adjusted depending upon the amount needed tocorrect defects in the subject's skin, or augment tissue in the subject.In particular, the concentration of “AUTOLOGEN” in the dispersion can beat least about 25 mg/L.

Another example of filler material comprises exogenous proteins, such asany type of collagen. Presently, numerous collagen products arecommercially available and have applications in the present invention.Examples of such products are reconstituted bovine collagen productscommercially available including, but not limited to, “ZYDERM I”,“ZYDERM II” and “ZYPLAST”, which comprise reconstituted bovine collagenfibers cross-linked with glutaraldehyde. These three products, whichhave been approved by the U.S. Food and Drug Administration (FDA) fortreating wrinkle lines and depressed scars since 1981, are produced bythe Collagen Corporation of Palo Alto, Calif.

Other examples of filler materials having applications in the presentinvention include, but are not limited to, solubilized gelatin,polyglycolic acid, or cat gut sutures. One particular is a gelatinmatrix implant sold under the mark “FIBRIL”, which comprises porcinegelatin powder plus o-aminocaproic acid, which are dispersed in a 0.9%(by volume) sodium chloride solution and an aliquot of plasma from thesubject, mixed in a 1:1 ratio by volume.

Methods for Correcting a Defect in Skin or Augmenting Tissue

Furthermore, the present invention extends to methods for correcting adefect in skin of a subject, such as scars, wrinkles or any of the otherskin, bone or subcutaneous defects, disorders, or diseases listedherein. It can be used for augmenting tissue in the subject,particularly facial tissue, e.g., augmenting the tissue of lips to makethe lips appear fuller. One such method of the present inventioncomprises injecting an effective amount of an injectable compositioncomprising autologous fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins) and abiodegradable, acellular injectable filler material, into the subject atthe site of the skin defect or desired tissue augmentation so thatregeneration of tissue at the site is promoted.

Another method encompassed by the present invention comprises the stepsof injecting passaged autologous fibroblasts substantially free ofimmunogenic proteins (e.g., culture medium serum-derived proteins) intothe subject at a site of a skin defect or desired tissue augmentation,and injecting a biodegradable, acellular injectable filler material intothe site. Preferably, the duration between injecting the autologousfibroblasts into the subject and injecting the biodegradable acellularinjectable filler into the subject is about two weeks.

Passaged, autologous fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins) can be readilyobtained using procedures set forth above. Furthermore, the varioustypes of biodegradable, acellular filler materials, which have beendescribed in detail above, have applications in the methods describedherein.

Injections set forth in the present invention are typically carried outwith a hypodermic syringe having a syringe chamber, a piston disposedtherein, an orifice communicating with the chamber, and a hypodermicneedle is fixed to the orifice. The size of the needle used in a methodfor correcting a defect in skin of a subject or augmenting tissue in thesubject ranges from approximately 30 gauge to approximately 27 gauge.With more viscous compositions needles of, e.g., 14 to 16 gauge can beused.

Initially, the tissue to be injected is prepped with alcohol andstretched to give a taut surface. After the tissue to be injected hasbeen prepped, a syringe is filled with an injectable composition of thepresent invention, if the first method described above is to be used, orwith passaged, autologous fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins) if the secondmethod for correcting skin defects or augmenting tissue is used. Theneedle is inserted into the tissue as superficially as possible, and theorientation of the bevel is not critical to the success of this methodof present invention. The actual injection is made by gentle pressureuntil a slight blanch is seen in the injected tissue. Multiple serialinjections can be made. Approximately two weeks after passagedautologous fibroblasts are injected into the subject's skin, an equalvolume of biodegradable, acellular filler is injected using the sameprocedure as described above into the same location where the passagedautologous fibroblasts were previously injected.

The present invention is not to be limited in scope by the specificembodiments described above, which are intended as illustrations ofaspects of the invention. Here functionally equivalent methods andcomponents are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description. Such modifications are intended to fall withinthe scope of the appended claims. Moreover, all cited references are,hereby, incorporated by reference.

1. A method of repairing bone tissue in a subject with a bone defect,wherein said method comprises: a) providing a composition, wherein thecomposition comprises (i) a biodegradable acellular matrix and (ii)autologous fibroblasts, wherein the composition is substantially free ofproteins derived from xenogeneic serum of xenogeneic serum-containingculture medium, wherein said biodegradable acellular matrix, prior tocombination with said autologous fibroblasts, comprises one or moresubstances selected from the group consisting of collagen,glycosaminoglycans, gelatin, polyglycolic acid, cat gut, demineralizedbone, hydroxyapatite, and anorganic bone, and wherein and saidautologous fibroblasts are added to the matrix at high density and thencultured for up to 4 days, such that sufficient autologous fibroblastsintegrate within said biodegradable acellular matrix to substantiallyfill the space on and within said biodegradable acellular matrixavailable for cells; b) identifying a site of a bone defect in saidsubject; and c) placing the composition on the site so that said bonedefect is repaired.
 2. The method of claim 1, wherein the bone defect isa dental ridge defect.
 3. The method of claim 1, wherein the bone defectis a defect of a mandibular bone.
 4. The method of claim 1, wherein thebone defect is a defect of a maxillary bone.
 5. The method of claim 1,wherein the bone defect is a defect of a bone selected from the groupconsisting of an orbital bone, a zygomatic bone, a cranial bone, or anasal bone.
 6. The method of claim 1, wherein the bone defect is a sinusfloor defect.
 7. The method of claim 1, wherein the bone defect is anon-union defect.
 8. The method of claim 7, wherein the non-union defectis a non-union defect of a long bone.
 9. The method of claim 1, whereinsaid biodegradable matrix, prior to combination with said autologousfibroblasts, comprises collagen and glycosaminoglycans, the collagenbeing cross-linked to the glycosaminoglycans with glutaraldehyde. 10.The method of claim 1, wherein said one or more substances are selectedfrom the group consisting of gelatin, polyglycolic acid, cat gutsutures, demineralized bone, and hydroxyapatite.
 11. The method of claim1, wherein said autologous fibroblasts are from gums, palate, or skin ofsaid subject.
 12. The method of claim 1, wherein said one or moresubstances comprise anorganic bone.
 13. The method of claim 12, said oneor more substances further comprising collagen.
 14. The method of claim1, wherein the collagen is bovine collagen.
 15. The method of claim 1,wherein the collagen is porcine collagen type I or porcine collagen typeIII.
 16. The method of claim 1, wherein said autologous fibroblasts,after being added to the matrix at high density, are cultured for 3 to 4days.